Alpha-amylase determinations are being resorted to with increasing frequency in clinical laboratories as an aid in diagnosing pancreatic and other diseases which alter normal concentration of alpha-amylase in a body fluid. Alpha-amylase is the common traditional name for the enzyme more precisely designated as alpha-1,4-glucan 4-glucanohydrolase.
Alpha-amylase plays a vital physiological role in enabling the digestion of starch and other polysaccharides by virtue of its specific enzymatic activity in the hydrolysis of alpha-1,4 linkages in the polysaccharide molecule, with the formation of maltose and various other oligosaccharides as ultimate products of the enzymatic reaction. Detection of abnormalities in the alpha-amylase content of body fluid provides an important aid in clinical diagnosis.
A number of analytical methods for the determination of alpha-amylase have been developed based upon the ability of alpha-amylase to hydrolyze starch. In all of these methods, starch or a starch derivative is digested with the sample containing alpha-amylase to be analyzed. Then the extent of the alpha-amylase induced degradation is measured by a variety of procedures. For example, the decrease in the amount of the starch substrate has been directly measured by an amyloclastic method, in which the decreased starch content is determined by adding iodine to the sample, and measuring the intensity of the blue colored starch-iodine complex which is formed. Alternatively, the decrease in starch content has been measured by the decrease in turbidity of an aqueous starch suspension following alpha-amylase digestion. Another chemical method for determining alpha-amylase activity involves use of a modified starch as the substrate, wherein the starch has a chromophore covalently linked to it. On digestion with alpha-amylase, small, water soluble colored fragments are formed, and the intensity of the color in the solution is measured photometrically.
Still another chemical analytical procedure is based upon reduction-oxidation analysis for reducing ends which are formed by the reaction of alpha-amylase with the starch substrate.
None of these chemical methods had proven entirely satisfactory for clinical use. All are time consuming, and require standard curves, while most also require the use of blanks, and long incubation times. Other problems include variability depending upon the source of the starch, sensitivities due to differences in laboratory technique and the need for specialized equipment.
Still other analyses are based upon enzymatic detection methods and, to date, all prior commercially available enzymatic alpha-amylase kits have been based upon measurements of the rate at which maltose is formed by the action of alpha-amylase on starch or an oligosaccharide substrate, such as maltotetraose or maltopentose. In one such analysis, maltose is converted to glucose by the enzyme alpha-glucosidase, and the level of glucose is in turn determined by conversion to glucose-6-phosphate by hexokinase, and conversion of glucose-6-phosphate to 6-phosphogluconate by reaction with beta-nicotinamide-adenine-dinucleotide (NAD) in the presence of glucose-6-phosphate dehydrogenase, as represented by the following sequence: ##STR1##
In this reaction system the conventional abbreviation NAD is used and is used elsewhere herein and in the claims, in reference to the coenzyme beta-nicotinamide-adenine-dinucleotide and NADH is used in reference to the same coenzyme in its reduced form. It also is to be understood that the NAD may be replaced by or used in admixture with NADP, namely, betanicotinamide-adenine-dinucleotide phosphate which in its reduced form is indicated as NADPH.
Still other procedures detect the formation of maltose via the enzyme maltose phosphorylase, as is illustrated by the following sequence: ##STR2##
Typically, the increase in absorbence at 340 nm due to increased levels of NADH is measured photometrically. These methods still are not as accurate or as sensitive as may be desired.
Still more recently, it has been proposed to use as the substrate for the alpha-amylase a starch which has been subjected to partial oxidation, with resulting damaging random effect on alpha-1,4 linked glucoses of the starch molecule to form a so-called "blocked" starch. In such a "blocked starch", the reactivity of the damaged alpha-1,4 linkages in blocked to sufficient extent to inhibit the activity of an exocarbohydrase on the starch molecule, while at the same time leaving residual alpha-1,4 linkages that are subject to alpha-amylase attack with attendant liberation of free non-reducing chain terminals that can be attacked by an exocarbohydrase, such as phosphorylase, which serves as a coupling enzyme in the following enzymatic reaction system: ##STR3##
The use of "blocked" starch in providing a substrate comprised in a coupled enzymatic reaction system is attended with inherent disadvantages in that, in producing the blocked starch substrate, the oxidation of the starch is only partial and damages alpha-1,4 linkages randomly throughout the starch molecule. As a result, difficulties are encountered as regards controlling the uniformity of the substrate and its reactivity with alpha-amylase and, as a corollary, correctly indicating the rate of change in absorbency occasioned by alpha-amylase in a body fluid. Moreover, because of the reduced overall activity occasioned by the partial oxidation, the amount of the "blocked" starch required for accomplishing a given change in absorbence is undesirably large and substantially interferes with the sensitivity of the test determination.